Enzyme variants with improved ester synthase properties

Patent metadata

Field	Value
Publication number	US-9879239-B2
Application number	US-201314424387-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Sep 14, 2012
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The disclosure relates to enzyme variants with improved ester synthase properties for the production of fatty acid esters. Further contemplated are recombinant host cells that express such variants, cell cultures comprising the recombinant host cells and fatty acid ester compositions produced by such recombinant host cells.

First claim

We claim:

1. A variant ester synthase polypeptide comprising a variant of SEQ ID NO: 67, wherein the variant of SEQ ID NO: 67 is genetically engineered to have an amino acid substitution at position 15 of SEQ ID NO: 67, and wherein said polypeptide comprises at least one additional mutation selected from the group consisting of G4R, T5P, T5S, D7N, T24M,T24W, L3OH, G33D, G33S, L39A, L39M, L39S, R40S, D41A, D41G, D41H, D41Y, V43K, V43 5 , T44A, T44F, T44K, Y58C, Y58L, A73Q, V76L, D77A, K78F, K78W, 180V, R98D, E99Q, G101L, I102R, P111D, P111G, P111S, H122S, R131M, I146K, I146E, I146R, S147A, V149L, R150P, V155G, T157S, R162E, N164D, N164R, P166S, T170R, T 1705 , V171E, V171F, V171H, V171R, V171W, R172S, R172W, P173W, R177V, A179S, A179V, D182G, E184F, E184G, E184L, E184R, E184S, A185L, A185M, S186T, V187G, V187R, P188R, A190P, A190R, A190W, S192A, S192L, S192V, Q193R, Q193S, M195G, A197T, A197V, Q201A, Q201V, Q201W, A202L, D203R, P206F, R207A, G212A, G212L, V216I, V219L, V234C, H239G, T242K, T242R, A243R, Q244G, R246A, R246G, R246L, R246Q, R246V, R246W, D255E, L257I, K258R, N259E, N259Q, L260V, H262Q, A263V, S264D, S264V, S264W, G265N, G266A, G266S, S267G, A285L, A285R, A285V, N288D, N289E, N289G, T293A, P294G, V301A, N302G, I303G, I303R, I303W, R304W, A306G, D307F, D307G, D307L, D307N, D307R, D307V, E309A, E309G, E309S, G310H, G310R, G310V, T311S, T313S, Q314G, I315F, S316G, F317W, I319G, A320C, A323G, D328F, Q334K, Q334S, Q348A, Q348R, K349A, K349C, K349H, K349Q, P351G, K352I, K352N, S353K, S353T, T356G, T356W, Q357V, M360Q, M360R, M360S, M360W, Y366G, Y366W, G375A, G375V, G375S, V381F, E393G, E393R, E393W, G394E, T395E, V409L, L411A, A413T, I420V, S424G, S424Q, S442E, S442G, M443G, A447C, A447I, A447L, L454V, D455E, L457Y, E458W, I461G, I461L, I461V, K466N, A468G, K472T and K472*, wherein said variant ester synthase polypeptide has improved fatty acid methyl ester synthase activity compared to a corresponding wild type ester synthase polypeptide.

2. The variant ester synthase polypeptide of claim 1 , wherein said polypeptide is from a Marinobacter hydrocarbonoclasticus (WS377) ester synthase polypeptide.

3. The variant ester synthase polypeptide of claim 1 , wherein expression of the variant ester synthase polypeptide in a recombinant host cell results in a higher titer of fatty ester compositions compared to a recombinant host cell expressing the corresponding wild type polypeptide.

4. The variant ester synthase of claim 3 , wherein said titer is at least about 5% or greater.

5. The variant ester synthase of claim 3 , wherein said fatty ester compositions are selected from the group consisting of fatty acid methyl esters, fatty acid ethyl esters, fatty acid propyl esters, fatty acid isopropyl esters, fatty acid butyl esters, monoglycerides, fatty acid isobutyl esters, fatty acid 2-butyl esters, and fatty acid tert-butyl esters.

6. The variant ester synthase of claim 1 , wherein said improved fatty acid methyl ester activity results in properties selected from the group consisting of increased beta hydroxy esters, decreased beta hydroxyl esters, increased chain lengths of fatty acid esters, and decreased chain lengths of fatty acid esters.

7. The variant ester synthase polypeptide of claim 1 , wherein expression of the variant ester synthase polypeptide in a recombinant host cell results in a production of an altered percentage of beta hydroxy esters as compared to a recombinant host cell expressing the corresponding wild type ester synthase polypeptide.

8. The variant ester synthase polypeptide of claim 7 , wherein said altered percentage of beta hydroxy esters is an increased or decreased percentage of beta hydroxy esters.

9. The variant ester synthase polypeptide of claim 1 , wherein the polypeptide comprises the mutation S15G.

10. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5S, P111S, V171R, P188R, F317W, S353T, V409L and S442G.

11. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5S, K78F, P111S, V171R, P188R, S192V, A243R, F317W, K349H, S353T, V409L and S442G.

12. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5S, V76L, P111S, V171R, P188R, K258R, S316G, F317W, S353T, M360R, V409L and S442G.

13. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5S, P111S, V171R, P188R, Q244G, S267G, G310V, F317W, A320C, S353T, Y366W, V409L and S442G.

14. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations P111S, V155G, P166S, V171R, P188R, F317W, Q348A, S353T, V381F, V409L and S442G.

15. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations L39S, D77A, P111S, V171R, P188R, T313S, F317W, Q348A, S353T, V381F, V409L, 1420V and S442G.

16. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations TSS, T24W, T44F, P111S, I146L, V171R, P188R, D307N, F317W, S353T, V409L and S442G.

17. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations TSS, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, Q348R, S353T, V381F, V409L and S442G.

18. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5S, K78F, P111S, V171R, P188R, S192V, R207A, A243R, D255E, L257I, N259Q, L260V, H262Q, G265N, A285V, N288D, N289G, F317W, H349K, S353T, V381F, V409L and S442G.

19. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5P, G33D, T44K, Y58L, P111S, R162E, V171R, P188R, R207A, V234C, Q244G, D255E, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, S442G and I461V.

20. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5P, G33D, T44K, Y58C, P111S, V171R, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353T, Y366W, G394E, V409L, A413T, S442G and I461V.

21. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5P, T44A, P111S, T157S, N164D, T170S, V171R, P188R, R207A, V216I, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, Q334K, S353T, Y366W, G394E, V409L, S442G and I461V.

22. The variant ester synthase polypeptide of claim 9 , wherein the ester synthase polypeptide comprises mutations T5P, Y58L, P111S, R162E, T170S, V171R, A179S, P188R, R207A, V234C, Q244G, L257I, S267G, D307N, G310V, F317W, A320C, S353K, Y366W, G394E, V409L, S442G and I461V.

23. A recombinant host cell genetically engineered to express the variant ester synthase polypeptide of claim 1 .

24. A cell culture comprising the recombinant host cell of claim 23 and a fatty ester composition.

25. The recombinant host cell of claim 23 , wherein the recombinant host cell produces a fatty ester composition with an altered percentage of beta hydroxy esters as compared to a host cell that expresses the wild type ester synthase polypeptide.

26. The recombinant host cell of claim 25 , wherein the altered percentage of beta hydroxy esters is an increased percentage of beta hydroxy esters.

Assignees

• Reg Life Sciences Llc
• Reg Life Sciences Llc

Inventors

• Shumaker Andrew M
• Da Costa Bernardo M
• Holden Kevin
• Hom Louis G
• Baron Tarah S
• Helman Noah

Classifications

• C12Y201/01015

  Fatty-acid O-methyltransferase (2.1.1.15) · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12P7/6436

  Fatty acid esters · CPC title

• Y02P30/20

  using bio-feedstock · CPC title